Histone deacetylase (HDAC) inhibitory and antiproliferative activities of phenolic-rich extracts derived from the rhizome of Hydnophytum formicarum Jack.: sinapinic acid acts as HDAC inhibitor

Dried rhizomes of H. formicarum Jack. were obtained from a local herbal shop in Khon Kaen Province, Thailand. The rhizomes were collected during March-May 2008, from Narathiwat Province, Thailand. Taxonomic identification was approved by the Forest Herbarium, Department of National Parks, Wildlife and Plant Conservation, Ministry of Natural Resources and Environment, Bangkok, Thailand. A voucher specimen (voucher number TS09001) is deposited at the KKU Herbarium, Department of Biology, Faculty of Science, Khon Kaen University, Khon Kaen, Thailand. Chemicals and most of the pure standards of phenolic acids were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). The pure standards of m-hydroxybenzaldehyde and p-hydroxybenzoic acid were purchased from Fluka (Buchs, Switzerland) and Acros Organics (Geel, Belgium), respectively.

Five grams of air-dried ground rhizome were macerated and periodically stirred in 50 ml of absolute ethanol for 48 hours. The suspension was filtered through Whatman No. 4 filter paper and centrifuged at 5,000 rpm for 15 minutes. The supernatant was air-dried to yield an ethanolic crude extract. The residue was reconstituted in dimethyl sulfoxide (DMSO) or ethanol before testing and the solvent was used as a negative control.

Five grams of air-dried ground rhizome were macerated and periodically stirred in 50 ml of hexane for 48 hours. The suspension was filtered through the filter paper and centrifuged at 5,000 rpm for 15 minutes. The supernatant was air-dried to obtain the hexane soluble fraction. The precipitate remaining from hexane extraction was dispersed, macerated and periodically stirred in 50 mL of ethyl acetate for 48 hours. The ethyl acetate suspension was filtered through the filter paper, centrifuged at 5,000 rpm for 15 minutes, and air-dried to obtain the ethyl acetate soluble fraction. The precipitate remaining from ethyl acetate extraction was dispersed, macerated and periodically stirred in 50 ml of methanol for 48 hours. The methanol suspension was filtered through the filter paper, centrifuged at 5,000 rpm for 15 minutes, and air-dried to obtain the methanol soluble fraction. Each solvent fraction was reconstituted in an appropriate vehicle, DMSO or ethanol, before testing.

Phenolic extraction was performed by using acidic hydrolysis method [20] with some modifications. Briefly, two hundred milliliters of 70% methanol were added to a beaker containing 10 grams of ground rhizome. The mixture was stirred for 2 hours at room temperature and then filtered through the filter paper. The filtrate was evaporated to 60 ml by a rotary evaporator. The remaining filtrate was added with 50 ml of 2 M NaOH and stirred continuously for 12 hours at room temperature. The mixture was centrifuged at 1,700 g for 20 minutes and then filtered through the filter paper. The supernatant was repeatedly extracted three times with 80 ml of diethyl ether, in which the aqueous phase was collected and the diethyl ether phase was discarded. The aqueous phase was adjusted to pH 1.5 by 10 M HCl and filtered through the filter paper. The filtrate was further extracted by 80 ml of diethyl ether for three times, in which the portion of the diethyl ether was collected. The pooled diethyl ether phase was dehydrated with sodium sulphate (Na₂SO₄) anhydrous and then filtered through the filter paper. The filtrate was evaporated to 5 ml using a rotary evaporator and finally evaporated to dryness under a gentle stream of nitrogen.

Total phenolic content in ethanolic crude extract was determined by the Folin-Ciocalteu method as described previously [21]. Gallic acid (Sigma) was used as the standard and the result was calculated as μg Gallic Acid Equivalent (GAE) per mg dry weight of the extract.

The identification of individual phenolic acids in phenolic-rich extract prepared by phenolic extraction as described above was carried out using a Waters HPLC system, based on matching spectrum and retention times of phenolic acid standards. The phenolic acid standards used were gallic acid, protocatechuic acid, p-hydroxybenzoic acid, vanillic acid, caffeic acid, syringic acid, m-hydroxybenzaldehyde, p-coumaric acid, ferulic acid, and sinapinic acid. The HPLC system consisted of a Waters 600E Multisolvent Delivery system, Waters In-Line degasser AF, a Rheodyne injector with sample loop of 20 μl (180 μg of the extract), and a Waters 2669 photodiode array (PDA) detector. Empower software was used for data acquisition. A Waters system column C18 (3.9 mm i.d. × 150 mm, 5 μm particle diameter) coupled to a guard column was used. The temperature of the column was 25°C and the flow rate of mobile phase was 1.0 ml/minute. The compounds were eluted with a gradient elution of mobile phase A (100% acetronitrile) and B (1% acetic acid in deionized water) where A increased from 3% to 8% in 5 minutes, to 10% by 25 minutes and was maintained at 10% for 20 minutes, then returned to initial condition (3%) in 10 minutes and remained for 5 minutes before next injection. Elutes were detected by the PDA detector at wavelength 280 nm.

HeLa and HT29 cells were obtained from the National Cancer Institute, Bangkok, Thailand. Jurkat cells were kindly provided by Dr. M. Leid (Oregon State University, Oregon, U.S.A.). HCT116 and MCF-7 cells were kindly provided by Dr. O. Tetsu (University of California, San Francisco, U.S.A.). Vero cells were kindly provided by Dr. S. Barusrux (Khon Kaen University, Khon Kaen, Thailand). Cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 μg/ml) (Gibco-BRL). The cells were incubated at 37°C in a humidified atmosphere with 5% CO₂.

Cells were seeded in a 96-well plate at cell density of 10⁴ cells/well and incubated for 24 hours. Sample groups were treated with different concentrations of H. formicarum Jack. rhizome extracts (0.5-3.0 mg/mL), sinapinic acid (0.5-3.0 mM), or sodium butyrate (0.5-3.0 mM) for 24, 48 and 72 hours. Vehicle control groups were added with DMSO (final concentration of 0.05%) or double distilled water. Cell proliferation assays were performed using a WST-8 Cell Proliferation Assay Kit (BioVision, Mountain View, CA, U.S.A.) according to the manufacturer’s instructions. Absorbance (A) was measured at 415 nm using a microtiter plate reader (Bio-Rad Laboratories, Hercules, CA, U.S.A.). The absorbance at 655 nm was used as a reference wavelength. Cell proliferation or cell growth was determined as a percentage of the vehicle control by an equation of;

Cells grown in a 4.5-cm dish were treated with either solvent control or the sample for 6 hours, and the histone proteins were then isolated according to the Abcam’s protocol (Abcam Inc., Cambridge, MA, U.S.A.) with some modifications. In brief, cells were harvested by trypsinization, washed with PBS, and then resuspended in Triton Extraction Buffer (TEB; PBS containing 0.5% (v/v) Triton X-100, 2 mM phenylmethylsulfonyl fluoride, 0.02% (w/v) NaN₃) at a cell density of 10⁵ cells/ml. The cells were incubated on ice and agitated periodically for 10 minutes. The suspension was centrifuged at 7,500 rpm for 10 minutes at 4°C to spin down the nuclei and the supernatant was discarded. The nuclei pellet was resuspended in 0.2 M HCl at a density of 10⁶ nuclei/ml and incubated overnight at 4°C. The suspension was centrifuged at 7,500 rpm for 10 minutes at 4°C and the supernatant containing histone proteins was collected. Protein concentration was measured by using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA, U.S.A.) based on the Bradford method.

Inhibition on acetylation of cellular histones was analyzed by gel electrophoresis using acid urea/Triton-X-100 (AUT) gels [22]. The upper gel consisted of 5% acrylamide/bis-acrylamide containing 0.9 M acetic acid, 8 M urea. The resolving gel was 15% acrylamide/bis-acrylamide containing 0.9 M acetic acid, 8 M urea, and 0.37% Triton X-100. The running buffer was 0.9 M acetic acid. In this buffer system, positively charged proteins migrate toward the cathode. Electrophoresis was performed in a Mini PAGE System (Select BioProducts, Edison, NJ, U.S.A.). Gels were pre-run at 150 volts for 4 hours at the ambient temperature. Wells were then loaded with the second pre-run solution (1 M cysteamine (Sigma-Aldrich), 8 M urea, 0.9 M acetic acid) to scavenge the residual free radicals and the gel was pre-run at 150 volts for a further 40 minutes. Histone samples (5 μg histone protein) solubilized in loading buffer (8 M urea, 10% glycerol, 0.9 M acetic acid, 5% β-mercaptoethanol and 0.25% methylene blue) were boiled for 5 minutes before being loaded and gels were run at 90 volts for 6 hours. Gels were silver-stained by using PageSilver™ Silver Staining Kit (Fermentas, Burlington, Ontario, Canada), dried, and photographed.

Apoptosis analysis was performed by using a Vybrant Apoptosis Assay Kit #2 (Molecular Probes, Invitrogen Corporation, Carlsbad, CA, U.S.A.) according to the manufacturer’s instructions. Briefly, cells were seeded at 1.2 × 10⁶ cells/4 ml in a 4.5-cm dish, incubated for 24 hours, and treated with different concentrations of the extracts or sinapinic acid for 6 hours. Cells were harvested by trypsinization, washed with cold PBS, and resuspended in the Annexin-binding buffer. Cell density was determined and diluted in the annexin-binding buffer to 10⁵ cells per assay. Cells were incubated with Alexa Fluor 488 Annexin V and Propidium iodide (PI) at room temperature for 15 minutes. Following the incubation, cells were analyzed by flow cytometry using a Beckman Coulter Cytomics FC500 MPL flow cytometry (Beckman Coulter, Miami, FL, U.S.A.). The flow cytometry results were confirmed by viewing the cells under a fluorescence microscope.

The effect of various polarity extracts including fractionated solvent extracts from hexane soluble fraction, ethyl acetate soluble fraction, methanol soluble fraction and also ethanolic crude extract on in vitro HDAC activity was examined by using HeLa nuclear extract as a source of the HDAC enzymes. As shown in Figure 1, all of the above mentioned extracts significantly inhibited HDAC activity (p < 0.05). Among various polarity extracts tested, ethanolic crude extract exhibited the most potent HDAC inhibition of 55.2 ± 3.2% as compared to the control. Therefore, this extract was used to investigate the further effects of this plant on cancer cells.

Several lines of evidence indicate that some plant phenolic compounds possess HDAC inhibitory activity [16, 18, 19]. Therefore, we intended to investigate the effect of phenolic extract from H. formicarum Jack. rhizome on HDAC activity in vitro. As expected, phenolic extract of this plant significantly inhibited HDAC activity (p < 0.05), and its effect was comparable to that of the ethanolic crude extract (Figure 1). The presence of phenolic compounds in the ethanolic crude extract was verified by the Folin-Ciocalteu reaction and total phenolic content was 316.28 ± 12.18 μg Gallic Acid Equivalent (GAE)/mg dry weight. Because phenolic-rich extract was found to possess HDAC inhibitory activity, therefore, this extract was also used to investigate the further effects on cancer cells.

Some phenolic compounds were previously discovered in the crude ethyl acetate extract of this plant [7], however, their HDAC inhibitory activity has not yet been explored. Preliminary separation and identification of individual phenolic compounds in phenolic extract was conducted by the reversed phase HPLC. Identification of sample peaks by matching against retention time and spectra of known phenolic standards under the same chromatographic conditions revealed that sinapinic acid was one of the two major components of phenolic-rich extract of H. formicarum Jack. rhizome (Figure 2A, 2B). The confirmation of peak was obtained by the addition of sinapinic acid standard into the sample for HPLC analysis (Figure 2C). The yield of phenolic-rich extract from 10 g of H. formicarum Jack. rhizome was 67.5 mg. The amount of sinapinic acid was 3.4 μg/mg of phenolic-rich extract. However, other sample peaks remained to be identified. Interestingly, sinapinic acid was found to act as HDAC inhibitor, blocking the enzyme activity in vitro with an IC₅₀ value (2.27 ± 0.13 mM) higher than that of the well-known HDAC inhibitor sodium butyrate (0.97 ± 0.07 mM) (Figure 3). These findings suggest that sinapinic acid takes account, at least in part, for the inhibition of HDAC activity by the plant phenolic extract (Figure 1).

HDAC inhibition by ethanolic crude extract, phenolic-rich extract and sinapinic acid in HeLa cells was analyzed by AUT gel electrophoresis, whereby each cellular core histone (H2A, H2B, H3, and H4) with different extent of acetylation can be separated. Herein, the profiles of histones H4 and H2B extracted from ethanolic crude extract-, phenolic-rich extract-, or sinapinic acid-treated HeLa cells were demonstrated (Figure 4). The addition of ethanolic crude extract and phenolic extract to cell cultures resulted in the accumulation of hyperacetylated histone H4 molecules, which could be detected clearly on AUT gel (Figure 4A). The histone H4 with three acetylated lysine residues was markedly increased when treated the cells with ethanolic and phenolic-rich extracts (Figure 4A). Similarly, treatment of HeLa cells with sinapinic acid clearly increased di- and tri-acetylated H4 molecules with two and three acetylated lysine residues, respectively (Figure 4B). However, HDAC inhibition of sinapinic acid in the cell was much less effective when compared to that of sodium butyrate. These observations indicated that ethanolic crude extract, phenolic-rich extract and sinapinic acid inhibited HDAC activity not only in vitro but also in the cells.

The anticancer activity of the two rhizome extracts and sinapinic acid was further investigated in 5 human cancer cell lines and in a non cancer cell line (Vero cells). As shown in Table 1, ethanolic and phenolic-rich extracts possessing HDAC inhibitory activity inhibited the growth of HeLa cells in a dose- and time-dependent manner with IC₅₀ values of 0.54 ± 0.03 and 0.30 ± 0.05 mg/ml, respectively, for exposure time of 72 hours. Phenolic-rich extract showed greater antiproliferative activity than ethanolic crude extract on growth inhibition of HeLa cells. However, both extracts showed no significant activity on non cancer cells and other cancer cell lines tested. Sinapinic acid significantly inhibited the growth of HeLa cells with an IC₅₀ value (0.91 ± 0.02 mM) lower than sodium butyrate (1.22 ± 0.03 mM) for exposure time of 72 hours. Sinapinic acid also showed greater antiproliferative activity than sodium butyrate on HT29 cells. The antiproliferative activity of sinapinic acid against HCT116 cells was not significantly different from that of sodium butyrate. In contrast, sinapinic acid showed a less efficient activity than sodium butyrate against Jurkat cells. Further, both sinapinic acid and sodium butyrate showed no significant activity on non cancer and breast cancer (MCF-7) cell lines. This finding suggests that sinapinic acid may underpin, at least in part, both the HDAC inhibitory activity and anticancer activity of the rhizome extracts.

Histone acetylation leads to modulation of expression of a specific set of genes that result in cell cycle arrest and induction of apoptosis [23, 24]. HDAC inhibitors induce apoptosis in a number of tumor cell types and through various mechanisms [25]. To investigate the mechanism of antiproliferative effect of ethanolic crude extract, phenolic extract and sinapinic acid on HeLa cells, we examined their capacity to induce apoptosis. Apparently, ethanolic crude extract, phenolic extract, and sinapinic acid exhibited a significant effect on induction of apoptosis in HeLa cells even only 6 hours of exposure time (Table 2). The treatment of HeLa cells with 1.4 mg/ml of ethanolic and phenolic-rich extracts resulted in the increase of early apoptotic cells up to 42.9% and 78.9%, respectively. The treatment with 9 mM of sodium butyrate and sinapinic acid resulted in the increase of early apoptotic cells up to 7.6% and 8.4%, respectively. In contrast, the control HeLa cells had only 0.95% of apoptotic cells. These results suggest that ethanolic crude extract and phenolic extract, as well as sinapinic acid, suppress the HeLa cell growth through induction of apoptosis.